Molecular differences between the human and pig erythrocyte ...

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BIOCHEMICAL SOCIETY TRANSACTIONS

Molecular differences between the human and pig erythrocyte nucleoside transporters FRANCIS Y. P. KWONG,* YUEN-MIN CHOY,* SIMON M. JARVISt and JAMES D. YOUNG* * Department qf' Biochemistry. Faculty of Medicine, The Chinese Unirersity of' Hong Kong. Shatin, N. T . , Hong Kong, and t Department qf Physiology. Unirersitv of'Alberta, Ednionton. Albertu. Cunuda T6G 2H7

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The entry of nucleosides into mammalian cells occurs largely by nucleoside-specific facilitated diffusion mechanisms which can be selectively inhibited by nanomolar concentrations of NBMPR (Young & Jarvis, 1983). Inhibition of nucleoside transport by NBMPR is associated with reversible high-affinity binding of inhibitor to the carrier mechanism (apparent K D 0.1-1 nM). This binding is normally reversible. However, exposure of site-bound [ 3H]NBMPR to high-intensity U.V. light results in the specific covalent radiolabelling of the transporter (Wu et at., 1983; Young et at., 1983; Shi et al., 1984). We report here a comparison of radiolabelled nucleoside transporters from human and pig erythrocytes. Human and pig erythrocyte membranes were photoaffinity labelled with [ 3H]NBMPR under equilibrium binding conditions in the absence and in the presence of excess non-radioactive NBTGR as described previously (Wu el at., 1983). The radiolabelled membranes were then subjected to SDS/polyacrylamide-gel electrophoresis by the method of Thompson & Maddy (1982). Gel-associated radioactivity was determined by slicing gels into 2mm fractions and measuring the 3H content of these slices by liquid scintillation counting (Wu et al., 1983). Fig. 1 compares the SDS/polyacrylamide-gel 3H profiles of radiolabelled human and pig erythrocyte membranes run on the same slab gel. Membranes from human species gave a major peak of radiolabelling in the band 4.5 region of the gel ( M , 45000-66000) [nomenclature of Steck (1974)l. However, the two radiolabelled transporters migrated with significantly different apparent M , values, the pig protein having the higher apparent M , . Covalent labelling of these polypeptides was abolished when photolysis was performed in the presence of NBTGR (data not shown). The minor high and low M , peaks in Fig. 1 represent aggregates of the transporters and degradation products, respectively. Both the human and pig erythrocyte membrane preparations exhibited non-specific labelling in the lipid region of the gel. The human and pig erythrocyte nucleoside transporters were also found to behave differently during DEAEcellulose ion-exchange chromatography of n-octyl glucoside membrane extracts. For these experiments, human and pig erythrocyte membranes were depleted of extrinsic membrane proteins and photoaffinity labelled with [3H]NBMPR as before except that photoactivation was performed in the absence of unbound 3H-labelled ligand (non-equilibrium binding conditions) to minimize the possibility of non-specific labelling (e.g. of membrane lipid) (Wu et al., 1983). Under these photolysis conditions, more than 80% of the covalently bound radioactivity represented specific radiolabelling of carrier protein. Samples were solubilized with 46m~-n-octylglucoside in 50m~-Tris/HCI, 2mM-dithiothreitol (pH 7.4 at 4°C) and the 13OOOOg supernatant applied to DEAE-cellulose columns equilibrated with the same buffer. Proteins bound to the columns were eluted with buffer containing 1 M-NaCI. For human erythrocyte membrane extracts, the majority of applied radioactivity (75%) was recovered in the column

This work was funded by a project grant from the Cancer Research Campaign, U . K . S. M. J . acknowledges support from the Medical Research Council. Canada, and the Alberta Heritage Foundation for Medical Research.

Abbreviationsused : NBMPR,nitrobenrylthioinosine;NBTGR. nitrobenzylthioguanosine;SDS. sodium dodecyl sulphate.

Jarvis, S. M. & Young, J . D. (1981) Biochem. J . 194, 331-339 Shi, M. M., Wu, J.-S. R., Lee, C. M. & Young, J . D. (1984) Biochem. Biophys. Res. Commun. 118, 594-600

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Fig. 1. Conipurison of'thephotoaffinity labelling qf'hunianurid pig erythrocyte nienihranes with [ HINBMPR Human (A)and pig erythrocyte 'ghosts' ( 0 ) were equilibrated with 75 nM-[ HJNBMPR, supplemented with dithiothreitol (final concentration 5 0 m ~ ) and , exposed to high-intensity U.V. light from a 450W mercury arc lamp (Wu et al., 1983) for 45s. Unreacted [3H]NBMPR was removed by washing and the washed membrane pellets (1OOpg of protein) electrophoresed in a 12% SDS/polyacrylamide-gel as described in the text. M , standards are from the same slab gel. The positions of the stacking gel-running gel interface and the tracking dye are indicated by A and B, respectively.

void-volume fractions containing only 9% of the applied protein. The 1 M-NaCI fractions contained large amounts of protein (mostly band 3), but little radioactivity. There was therefore an 8-fold purification of radiolabelled transporter during ion-exchange chromatography, a value similar to that obtained by Jarvis & Young (1981) using an assay based on reversible [ 3H]NBMPR-binding activity. In contrast, void-volume fractions from pig erythrocyte membrane extracts contained only traces of radioactivity. The majority of applied 3H and protein eluted in the 1 ~ NaCl fractions. We conclude from these experiments that there are significant molecular differences between the human and pig erythrocyte nucleoside transporters. Isolation techniques devised for the partial purification of the human erythrocyte transporter (Jarvis & Young, 1981) are not appropriate for the pig system.

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609th MEETING, LEEDS Steck, T. L. (1974) J . Cell Biol. 62, 1-19 Thompson, S. & Maddy, A. H . (1982) in Red Cell Membranes-A Methodolo~icalApproach(Ellory,J. C. &Young, J. D., eds.), pp. 67-94, Academic Press, London

239 Wu, J.-S. R., Kwong, F. Y. P., Jarvis, S. M. &Young, J . D. (1983) J . Biol. Chem. 258, 13745-13751 Young, J. D. & Jarvis, S . M. (1983) Biosci. Rep. 3, 309-322 Young, J. D., Jarvis, S. M., Robins, M. J . & Paterson, A. R . P. (1983) J . Biol. Chem. 258, 2202-2208

An investigation of transglutaminase activity during liver regeneration R. N. BARNES,* P. L. WALTON? arid M. GRIFFIN*

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of LIfk Sciences. Trent Polj technic, Clifton. Nottingham N G l l 8 N S , U . K . , and t I C 1 Pharmaceuticals, Alderley Park, Cheshire SKlO 4TJ, U . K .

The Ca?+-dependent enzyme transglutaminase (EC 2.3.2.13) catalyses the acyltransfer reaction between peptide bound glutamine residues and prima.ry amine groups, resulting in the incorporation, of amines into proteins or if the amine is peptide-bound lysine th.e cross-linking of proteins via c(y-glutamy1)-lysine bridges (Folk & Finlayson, 1977). The cellular function of the tissue transglutaminase is poorly understood but evidence suggests it may play an important role in the mediation of events at the cell membrane (Davies et al., 1980; Bungay et al., 1984). Studies on transglutaminase in neoplastic tissue have indicated lowered amounts of enzyme activity to be present, leading to the suggestion that rapidly proliferating tissue has a requirement for reduced levels of transglutaminase activity (Birckbichler et al., 1981). In hepatocellular carcinomas induced in rats it has been demonstrated that the lowering of transglutaminase activity is accompanied by a redistribution of the enzyme to the particulate fraction of the cell, suggesting that the cellular location of the enzyme in the liver cell may be important in the regulation of enzyme activity (Barnes et al., 1984). As a further control to this work the present study has measured the activity and distribution of transglutaminase in normal proliferating tissue, employing the regenerating ral liver as a model system Remington & Russell, 1982). Partial hepatectomy was undertaken as described by Higgins & Anderson (1931). in which approx. 70:gof the rat liver (from Wistar derived rats) was removed. Samples of regenerating liver were then removed at (days 3,8, 13 and 20. Sham-operated rats were used as controls. Cell proliferation was monitored by the incorporation of [3H]thymidine into liver DNA and was found to be consistent with the data of Lewan (rt al. (1977) with maximum incorporation occurring at day 3 (5.5-fold greater than controls), a 2-fold increase above controls at day 8 and equal to control levels at day 13 and d.iy 20. Samples of tissue to be assayed for transglutaminase activity were homogenized in a Potter-Hvejhem homogenizer in 0.25 M-sucrose/l rnM-Tris/HCI/l mM-EDTA buffer, pH 7.4, and the homogenate centrifuged at 600g for IOmin to give a nuclear pellet fraction (NP). The supernatant was then recentrifuged at 71000g for 45miri to give a particlefree supernatant (PFS) and the remaining particulate fraction (P) of the cell. Transglutaminase activity was measured by the Ca'+dependent incorporation of [ lJC]putrescine into NN'dimethylcasein (Lorand r t al., 1972). By using this protocol measurement of t,ransglutaminase in samples of homogenate obtained from the different time periods indicated little change in enzyme activity when expressed in terms of wet weight of tissue (Fig. 1). However when expressed in terms of DNA, an approximate measure of enzyme activity per cell, a 45% reduction in control levels ( P~ 0 . 0 5occurred ) at

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day 3, although no significant change was shown at days 8, 13 or 20. Measurement of the subcellular distribution of transglutaminase in the different samples (Fig. 1 ) indicated little change from that seen in control livers at day 3 and 20. However, at days 8 and I 3 an apparent redistribution of enzyme activity to the cytosol fraction of the cell ( P ~ 0 . 0 at 5 8 days) occurred which could not be accounted for by a change in redistribution of cellular protein.